Han Jie, Liu Bin, Tang Jingjing, Zhang Shuqin, Wang Xiaoshen, Li Xuzichao, Zhang Qian, Liu Zhikun, Wang Wanyao, Liu Yingcan, Zhou Ruimin, Yin Hang, Wei Yong, Li Zhuang, Zhang Minjie, Deng Zengqin, Zhang Heng
Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), State Key Laboratory of Experimental Hematology, Tianjin Medical University Cancer Institute and Hospital, The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Tianjin Institute of Immunology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin, China.
Tianjin Key Laboratory of Cellular Homeostasis and Disease, Department of Biochemistry and Molecular Biology, Tianjin Medical University, Tianjin, China.
EMBO J. 2025 Aug 20. doi: 10.1038/s44318-025-00544-8.
Defense-associated reverse transcriptase (DRT) systems are implicated in prokaryotic resistance to viral infections, yet the molecular mechanisms underlying their functionality remain largely unknown. Here, we characterize a two-component DRT9 system, composed of a reverse transcriptase (RT) and a non-coding RNA (ncRNA), which exhibits a protein-primed DNA synthesis activity upon phage infection. We also determine its cryo-electron microscopy (cryo-EM) structures in different functional states. DRT9 RT binds to ncRNA, forming a dimer of dimers configuration that assembles into a trimer of dimers upon substrate binding. This oligomerization transition, crucial for DRT9-mediated anti-phage defense, is facilitated by a ncRNA cooperative self-assembly manner. Furthermore, substrate binding induces large conformational movements around the catalytic pocket of DRT9 RT, revealing a "lock-switch" mechanism for enzymatic activation. Notably, phylogenetic analysis and functional assays identify a unique N-terminal helix extension required for ncRNA stabilization and enzymatic activity, distinct from previously reported reverse transcriptase systems. Overall, our findings illuminate the molecular basis of DRT9-mediated antiviral defense and expand the functional and mechanistic diversity of the DRT family.
防御相关逆转录酶（DRT）系统与原核生物对病毒感染的抗性有关，但其功能的分子机制在很大程度上仍不清楚。在这里，我们描述了一个由逆转录酶（RT）和非编码RNA（ncRNA）组成的双组分DRT9系统，该系统在噬菌体感染时表现出蛋白质引发的DNA合成活性。我们还确定了其在不同功能状态下的冷冻电子显微镜（cryo-EM）结构。DRT9 RT与ncRNA结合，形成二聚体的二聚体构型，在底物结合时组装成二聚体的三聚体。这种寡聚化转变对DRT9介导的抗噬菌体防御至关重要，由ncRNA协同自组装方式促进。此外，底物结合诱导DRT9 RT催化口袋周围的大构象运动，揭示了酶激活的“锁开关”机制。值得注意的是，系统发育分析和功能测定确定了ncRNA稳定和酶活性所需的独特N端螺旋延伸，这与先前报道的逆转录酶系统不同。总体而言，我们的发现阐明了DRT9介导的抗病毒防御的分子基础，并扩展了DRT家族的功能和机制多样性。
